α-突触核蛋白对酪氨酸羟化酶基因启动子的影响

目的 探讨α-突触核蛋白对多巴胺代谢的调节机制.方法 以人基因组DNA为模板,PCR法扩增酪氨酸羟化酶(Tyrosine Hydroxylase,TH)基因区的部分DNA片段(-495～ 25),将其插入质粒pGL3-Basic,构建荧光素酶报告基因重组质粒pGL3-THprom,转染多巴胺能神经元细胞系MES23.5和MES23.5/hα-Syn .结果 双荧光素酶活性分析表明,pGL3-Basic、pGL3-THprom和pGL3-Control在MES23.5细胞中表达的荧光素酶活性分别为5.60±0.67,26.80±4.11和32.90±4.75,而pGL3-THprom在MES23.5和MES23.5/hα-Syn 中表达的荧光素酶活性分别为26.80±4.11和14.40±0.61,差异极显著(P＜0.01).结论 这些结果提示扩增的DNA片段具有启动子活性,而α-Syn作为反式作用因子通过影响TH基因启动子的功能发挥对多巴胺代谢的负性调控作用.     

Lactacystin对大鼠纹状体DAT和VMAT2蛋白表达的影响

目的:探讨蛋白酶体抑制剂lactacystin对大鼠纹状体多巴胺转运体(DAT)和囊泡单胺转运体2(VMAT2)蛋白表达的影响。方法:在大鼠单侧黑质致密部立体定向分别注射lactacystin0.2、2、8μg,对照组注射等量的生理盐水。观察大鼠自主行为和阿扑吗啡诱导旋转行为的变化;应用免疫组化观察黑质细胞α-synuclein和纹状体DAT和VMAT2蛋白表达。结果:注射lactacystin0.2μg组未见明显异常;2μg组和8μg组出现进行性的运动迟缓、少动、震颤、头向健侧倾斜;注射阿扑吗啡后,8μg组大鼠出现向健侧旋转运动。随着lactacystin剂量的增加,2μg组和8μg组黑质细胞α-synuclein的表达分别较对照组高出57%(P<0.01)和122%(P<0.001);纹状体DAT和VMAT2的表达分别较对照组减少了40%(P<0.01)、85%(P<0.001)和50%(P<0.01)和88%(P<0.001);DAT和VMAT2的比值分别较对照组高出了18%和32%。结论:Lactacystin可能通过抑制DAT和VMAT2的表达导致黑质细胞变性坏死及帕金森病的发生。     

α-突触核蛋白对酪氨酸羟化酶基因启动子的影响(英文)

目的探讨α-突触核蛋白对多巴胺代谢的调节机制。方法以人基因组DNA为模板,PCR法扩增酪氨酸羟化酶(Tyrosine Hydroxylase, TH)基因区的部分DNA片段(-495~+25),将其插入质粒pGL3-Basic,构建荧光素酶报告基因重组质粒pGL3-THprom,转染多巴胺能神经元细胞系MES23.5和MES23.5/hα-Syn+。结果双荧光素酶活性分析表明,pGL3-Basic、pGL3-THprom和pGL3-Control在MES23.5细胞中表达的荧光素酶活性分别为5.60±0.67, 26.80±4.11和32.90±4.75,而pGL3-THprom在MES23.5和MES23.5/hα-Syn+中表达的荧光素酶活性分别为26.80±4.11和14.40±0.61,差异极显著(P<0.01)。结论这些结果提示扩增的DNA片段具有启动子活性,而α-Syn作为反式作用因子通过影响TH基因启动子的功能发挥对多巴胺代谢的负性调控作用。     

人酪氨酸羟化酶基因真核表达载体的构建及在哺乳动物细胞中的表达

用限制性内切酶EcoRI从pKS(-)HTH_1切下全长为1.9 kb的人酪氨酸羟化酶基因,在T_4DNA连接酶的作用下连接在真核表达载体pCDNA_3的EcoR Ⅰ位点,构建成重组质粒pcD-NA_3HTH_1,该质粒转染COS-7细胞,免疫荧光组织化学染色证实酪氨酸羟化酶在其中的表达。     

酪氨酸羟化酶与帕金森病

酪氨酸羟化酶 (tyrosinehydroxylase ,TH)是脑内多巴胺 (dopamine ,DA)合成的限速酶 ,在以黑质纹状体DA系统功能不足为主要表现的帕金森病 (Parkinson’sdisease ,PD )中 ,TH不仅作为继发因素产生一系列异常改变 ,而且由于其可以产生活性氧类 (reactiveoxygenspecies ,ROS)并可为ROS攻击失活 ,故而可能是一个重要的原发因素直接参与了PD发病。这为PD与TH关系的研究提供了一个新的重要视点。     

急性鱼藤酮中毒大鼠α-突触核蛋白与酪氨酸羟化酶的表达

目的探讨鱼藤酮中毒大鼠α-突触核蛋白与酪氨酸羟化酶免疫活性的变化。方法采用大鼠背部皮下持续注射小剂量鱼藤酮的方法,观察大鼠运动功能和生物学特征的变化。结果接受鱼藤酮注射的大鼠均出现明显的运动功能障碍,最严重表现是运动不能。大部分大鼠的黑质-纹状体酪氨酸羟化酶阳性神经元数目减少,全部大鼠的黑质细胞内均有α-突触核蛋白的过度表达。结论鱼藤酮中毒大鼠可以产生帕金森病的运动障碍及有关病理生理学特征。     

酪氨酸羟化酶与帕金森病

酪氨酸羟化酶是左旋多巴递质合成中的一个限速酶 ,在帕金森病中起重要作用。综述了酪氨酸羟化酶 (TH)的性质和结构特点 ,调节机制以及在帕金森病治疗中的作用。     

死亡相关蛋白小分子干扰RNA对帕金森病大鼠黑质酪氨酸羟化酶表达的影响

目的:利用动物模型探讨死亡相关蛋白(FADD)小分子干扰RNA(siRNA)对帕金森病(PD)大鼠黑质酪氨酸羟化酶(TH)表达的影响。方法:40只雄性Wistar大鼠随机分为4组。经立体定向定位黑质,PD组:注入6-羟基多巴(6-OHDA);siRNA组:注入FADD siRNA2次后再注入6-OHDA;SiRNA阴性对照组:注入FADD siRNA阴性对照物2次后再注入6-OHDA;正常对照组:注入抗坏血酸生理盐水。各组大鼠术后4周处死,取鼠的术侧中脑黑质,利用原位杂交检测FADD、TH mRNA表达。结果:PD组与正常对照组比较FADD mRNA表达显著增加(P<0.01);siRNA组与PD组比较FADD mRNA表达显著减少(P<0.05);siRNA阴性对照组与正常对照组比较FADD mRNA表达量无显著差异(P>0.05)。PD组与正常对照组比较TH mRNA表达显著减少(P<0.01);siRNA组与PD组比较TH mRNA表达显著增加(P<0.05);siRNA阴性对照组与正常对照组比较TH mRNA表达量差异无统计学意义(P>0.05)。结论:FADDsiRNA可能通过抑制凋亡对PD大鼠模型有一定的保护和治疗作用。     

Nurr1在MN9D细胞中的过表达及其对酪氨酸羟化酶的影响

目的:观察MN9D细胞中Nurr1的表达和分布及Nurr1过表达对酪氨酸羟化酶的影响.方法:应用高压液相色谱、免疫细胞化学、转基因、Western blot和Northern blot等方法检测了MN9D细胞中多巴胺及其代谢物的水平和Nurr1蛋白在细胞中的分布,建立了过表达Nurr1的细胞株并观察了Nurr1过表达对TH基因的影响.结果:MN9D细胞裂解液中含有大量多巴胺及其代谢物;Nurr1蛋白主要分布在MN9D细胞的胞质中,尤以核周最多;MN9D细胞中有Nurr1的mRNA存在和蛋白质的表达;过表达Nurr1的MN9D细胞A1株酪氨酸羟化酶表达增高.结论:Nurr1与多巴胺能神经元发育有关,有可能用于帕金森病的基因治疗.     

神经干细胞转染酪氨酸羟化酶基因后的分化

目的 探讨神经干细胞（NSCs）转梁酪氨酸羟化酶（TH）基因后的分化。方法 从胚胎16天Wistar大鼠室管膜前下周围区分离、增殖、鉴定NSCs；将TH基因和缺陷性逆转录病毒载体N2A构建成真核表达质粒，以电穿孔将其转入PA317包装细胞内；收集PA317包装产生的逆转录病毒颗粒，感染体外培养的NSCs，经G418筛选，获得成功转染TH基因的NSCs克隆；分别以0.4ng/ml bFGF和5％胎牛血清诱导转染及未转染TH基因的NSCs分化，比较TH基因转染及不同诱导方式对NSCs分化的影响，同时在分化细胞内检测TH的表达。结果 以0.4ng/ml bFGF诱导可使95％以上的NSCs分化为神经元，而5％FBS诱导则大多分化为神经胶质细胞，无论是否转染TH基因，神经元及神经胶质细胞的分化比例不成生改变；TH基因转染后能在神经干细胞的子代细胞内高效、稳定表达。结论 TH基因转染不影响NSCs的分化潜能，TH能在神经干细胞的子代细胞中有效表达0.4ng/ml bFGF诱导可以促使NSCs分化为神经元。     

Association between Antipsychotics-Induced Restless Legs Syndrome and Tyrosine Hydroxylase Gene Polymorphism

Objective

Restless legs syndrome (RLS) has been reported to be more prevalent in schizophrenic patients who take antipsychotics. The cause of RLS is unknown but associated with dopaminergic deficiency. Tyrosine hydroxylase (TH) is the enzyme responsible for catalyzing the conversion of L-tyrosine to DOPA. The purpose of this study is to determine whether the TH gene Val81Met polymorphism is associated with antipsychotic-induced RLS.

Methods

One hundred ninety Korean schizophrenic patients were evaluated by the diagnostic criteria of the International RLS Study Group (IRLSSG). The genotyping was performed by PCR-based methods.

Results

Of the one hundred ninety schizophrenic patients, 44 (23.2%) were found to have RLS. Although there were no significant associations between TH genotypes or allele frequencies and RLS, when separate analyses were performed by sex (male or female), we detected significant differences in the frequencies of the genotype (χ²=6.15, p=0.046) and allele (χ²=4.67, p=0.031) of the TH gene Val81Met polymorphism between those with and without RLS in the female patients.

Conclusion

These findings suggest that the TH gene Val81Met SNP might be associated with antipsychotic-induced RLS in female schizophrenic patients.     

Tyrosine Hydroxylase Replacement in Experimental Parkinson's Disease with Transvascular Gene Therapy

Summary: Transvascular gene therapy of Parkinson''s disease (PD) is a new approach to the gene therapy of PD and involves the global distribution of a therapeutic gene to brain after an intravenous administration and transport across the blood-brain barrier (BBB). This is enabled with the development of a nonviral gene transfer technology that encapsulates plasmid DNA inside pegylated immunoliposomes or PILs. An 85- to 100-nm liposome carries the DNA inside the nanocontainer, and the liposome surface is conjugated with several thousand strands of 2000-Da polyethyleneglycol (PEG). This PEGylation of the liposome stabilizes the structure in the blood stream. The liposome is targeted across the BBB via attachment to the tips of 1-2% of the PEG strands of a receptor-specific monoclonal antibody (mAb) directed at a BBB receptor, such as the insulin receptor or transferrin receptor (TfR). Owing to the expression of the insulin receptor or the TfR on both the BBB and the neuronal plasma membrane, the PIL is able to reach the neuronal nuclear compartment from the circulation. Brain-specific expression is possible with the combined use of the PIL gene transfer technology and brain-specific gene promoters. In the 6-hydroxydopamine rat model of experimental PD, striatal tyrosine hydroxylase (TH) activity is completely normalized after an intravenous administration of TfRmAb-targeted PILs carrying a TH expression plasmid. A treatment for PD may be possible with dual gene therapy that seeks both to replace striatal TH gene expression with TH gene therapy, and to halt or reverse neurodegeneration of the nigro-striatal tract with neurotrophin gene therapy.     

Dieldrin对PC12细胞的增殖及酪氨酸羧化酶mRNA表达的影响

目的 :观察有机氯杀虫剂dieldrin对培养的PC12细胞增殖及对其酪氨酸羟化酶mRNA(THmRNA)表达的影响 ,探讨该毒物致帕金森病 (PD)的可能机制。方法 :以MPP+ 为阳性对照 ,用不同浓度 (10 -4 ～ 1mmol·L-1)的MPP+ 和dieldrin分别于预PC12细胞 2 4h后计细胞数 ,并采用半定量逆转录聚合酶链式反应 (RT PCR)方法测定THmRNA表达的改变。结果 :(1)MPP+ 和dieldrin对PC12细胞均有毒性 ,1mmol·L-1的MPP+ 可使细胞数减少至对照组的 45 % (P <0 0 1) ,低于此浓度的MPP+ 对细胞增殖无影响 ;但 0 1mmol·L-1的dieldrin就可使细胞数减少至对照组的 3 0 % (P <0 0 1) ,1mmol·L-1的dieldrin使PC12细胞全部死亡。 (2 )MPP+ 和dieldrin均可降低THmRNA的表达。 0 1mmol·L-1MPP+ 和dieldrin使细胞THmRNA的表达分别下降至对照组的 69 3 % (P >0 0 5 )和 61 7% (P <0 0 1)。结论 :Dieldrin不仅具有与MPP+ 相同的抑制培养的PC12细胞的增殖及THmRNA表达的作用 ,而且其对细胞的毒性强于MPP+ ,提示dieldrin是一种潜在的致PD物质 ,其与PD的关系值得进一步研究     

表皮生长因子受体及酪氨酸激酶产物在脑膜瘤中的表达

目的 检测脑膜瘤中表皮生长因子受体及其相关癌基因蛋白的表达,探讨酪氨酸激酶信号传导在脑膜瘤发展中的意义,为酪氨酸激酶抑制剂治疗肿瘤提供依据。方法 用免疫组化SABC法检测了20例脑膜瘤中EGFR、 erbB2蛋白及磷酸酪氨酸（P－Tyr)的表达,分析它们与脑膜瘤病理级别的关系。结果 20例脑膜瘤中均有EGFR、erbB2,P-Tyr在肿瘤细胞及瘤中血管内皮细胞上的不同程度表达,但以间变型脑膜瘤中表达最多,在纤维型与合体细胞型的表达无显性差异。结论 脑膜瘤中有EGF活性受体及erbB2蛋白的表达。它们可能与某些脑膜瘤的恶性行为有关,酪氨酸激酶信号传导在脑膜瘤发展中可能有重大意义。     

2004/低频脉冲电场对PC12细胞酪氨酸羟化酶和多巴胺合成的影响

运用Western 印迹和HPLC分别测定不同时间电场刺激和刺激后不同培养时间条件下,PC12细胞内酪氨酸羟化酶（TH）和细胞培养液中多巴胺（DA）含量的变化。结果显示,受到短时间（5、10 min）脉冲电场刺激的PC12 细胞,经较短时间（2天）的培养后,细胞内TH的含量和培养液中DA的含量均比对照组有所提高,但随着培养时间的延长（3~5天）,TH和DA的含量均明显下降。然而,长时间（15、20、30 min）脉冲电场刺激组则先表现为TH和DA的合成受到抑制,但随着培养时间的延长,其合成则被逐渐激活。采用蛋白激酶A（PKA）特异性抑制剂H-89和有丝分裂原活化蛋白激酶的激酶（MEK1/2）特异性抑制剂U0126,研究脉冲电场刺激所激活的与TH和DA合成相关的信号通路。结果表明,在没有神经生长因子（NGF）存在的情况下,PC12细胞主要通过PKA通路来激活TH的合成,低频脉冲电场刺激也主要激活PKA通路,因为抑制这条信号通路能显著抑制电场刺激所诱导的TH合成。     

Comparison of the Temporal Programs Regulating Tyrosine Hydroxylase and Enkephalin Expressions in TIDA Neurons of Lactating Rats Following Pup Removal and then Pup Return

Dopamine (DA) and enkephalin (ENK) release from the tuberoinfundibular dopaminergic neurons (TIDA) into the hypophysial portal circulation is fundamentally different under non-lactating and lactating conditions. The aim of this experiment was to compare the effect of a brief interruption then resumption of suckling on the temporal program of tyrosine hydroxylase (TH; rate-limiting enzyme of dopamine synthesis) and ENK regulation in dams. On post partum day 10, pups were removed for a 4-h period from a group of the dams then returned for 4- and 24-h periods. It was examined whether such a brief interruption of suckling provokes full up-regulation of TH and down-regulation of ENK, and whether reinitiation of suckling limits the extent to which TH up- and ENK down-regulate. At the end of experiment, the animals were decapitated. In situ hybridization was used to examine the expression of TH and ENK mRNA in the arcuate nucleus where TIDA neurons reside. The results showed that, on one hand, the removal of pups induced TH up-regulation, on the other hand, ENK expression also increased 8 h after removal of pups and then started to slowly decline. In dams whose sucklings were reinitiated both TH and ENK mRNAs were up-regulated at least for a day. ENK expression responded more slowly to the removal of pups than expression of TH, and after reinitiation of suckling, the temporal program of regulation of both TH and ENK expressions ran parallel in the first 24 h.     

Lithium Chloride Induces the Expression of Tyrosine Hydroxylase in hNT Neurons

In the present study, several doses of lithium chloride were tested for their ability to induce the expression of tyrosine hydroxylase (TH) in neurons derived from a human teratocarcinoma cell line (hNT) after 5 and 10 days in vitro (DIV). Following immunocytochemical staining for tyrosine hydroxylase, the percentage of TH-positive neurons was determined and morphometric analysis, including mean soma profile area and neuritic length, was performed. hNT neurons responded to lithium treatment in a dose-dependent manner. In 5 DIV, the most effective dose of lithium chloride (1.0 mM) increased the number of TH-positive neurons approximately sixfold. In addition, both TH-positive hNT neuron mean soma profile area and neurite length were significantly larger than controls by 60 and 70%, respectively. Moreover, even after withdrawal of lithium chloride on day 5, the number of TH-positive neurons in 10 DIV cultures remained significantly increased. These data suggest that hNT cells are indeed responsive to lithium exposure and may serve as a continual source of TH-expressing neurons in new therapeutic approaches to degenerative brain disease.     

Effects of Dehydroepiandrosterone (DHEA) on Pituitary Prolactin and Arcuate Nucleus Neuron Tyrosine Hydroxylase mRNA Levels in the Rat

It is well documented that dehydroepiandrosterone (DHEA), an adrenal androgen, is converted into potent androgens and/or estrogens in peripheral tissues. Since sex steroids are involved in the regulation of prolactin (PRL) secretion, we have studied the effect of DHEA administration on PRL mRNA levels in both adult male and female rats. Since tuberoinfundibular dopaminergic (TIDA) neurons are involved in the negative regulation of PRL, we have also evaluated the effects of DHEA on the genetic expression of tyrosine hydroxylase (TH), the limiting enzyme in catecholamine biosynthesis in TIDA neurons. Sham-operated and castrated animals of both sexes received during 2 days DHEA at the dose of 6 mg/kg/day, starting on the first day after castration. PRL and TH mRNA levels were measured by quantitative in situ hybridization. In the male rat, orchiectomy performed 3 days earlier did not modify PRL mRNA levels. DHEA administration increased the hybridization signal in both sham-operated and orchiectomized animals. In the female, ovariectomy decreased PRL mRNA levels and, as observed in the male, DHEA treatment induced an increase in the hybridization signal in both control and ovariectomized rats. In TIDA neurons, castration increased TH mRNA levels as evaluated by number of grains over labelled neurons and the number of TH-labelled cells per section in both male and female animals. In both sham-operated male rats and orchiectomized animals, DHEA decreased the hybridization signal. In the female, DHEA administration completely prevented the increase in TH mRNA levels due to ovariectomy. In sham-operated female rats, the treatment had no effect. These data clearly indicate that in both male and female rats DHEA exerts an estrogenic influence on both PRL and TH gene expression. Although these in vivo experiments do not allow to establish whether the stimulation of PRL gene expression is due to an action of the steroid on the pituitary or at the hypothalamic level or alternatively at both sites, it is likely that one of the mechanisms of action of DHEA might be related to a decrease in dopamine release following a depression of TIDA neuron activity.